The invention is in the field of models for medical diseases. Specifically, the invention is in the field of neurodegenerative disease models, and especially, Alzheimer""s disease models.
As human life span has significantly expanded over the last century, Alzheimer""s disease and other neurodegenerative diseases will have a growing impact on the quality of life for a large proportion of the population. For example, Alzheimer""s disease is a leading cause of dementia in the elderly, affecting 5-10% of the population over the age of 65 years. See A Guide to Understanding Alzheimer""s disease and Related Disorders, edited by Jorm, New York University Press, New York (1987). Alzheimer""s disease often presents with a subtle onset of memory loss followed by a slow progressive dementia over several years. The prevalence of Alzheimer""s disease and other dementias doubles every five years beyond the age of 65. See 1997 Progress Report on Alzheimer""s disease, National Institute on Aging/National Institute of Health. Alzheimer""s disease now affects 12 million people around the world, and it is projected to increase to 22 million by 2025 and to 45 million by 2050. See Alzheimer""s Association Press Release, Jul. 18, 2000.
The complexity of the brain""s architecture and chemistry, and the complexity of these neurodegenerative brain diseases, especially Alzheimer""s disease, has hampered the development of a model that mimics many of the changes seen in the human brain. Such a model is needed in order to identify drugs or other agents that might be useful in treating, preventing or reversing the effects of such diseases.
Alzheimer""s disease is histopathologically characterized by the loss of particular groups of neurons and the appearance of two principal lesions within the brain, termed senile plaques and neurofibrillary tangles. See Brion et al., J. Neurochem. 60:1372-1382 (1993). Senile plaques occur in the extracellular space. A major component of senile plaques is beta-amyloid (A-beta), a naturally secreted but insoluble peptide formed by cleavage of amyloid precursor protein (APP). A-beta is a fragment close to the carboxyterminal domain of APP.
Neurofibrillary tangles are intraneuronal accumulations of filamentous material in the form of loops, coils or tangled masses. They are most abundantly present in parts of the brain associated with memory functions, such as the hippocampus and adjacent parts of the temporal lobe. See Robbins Pathologic Basis of Disease, Cotran et al., 6th ed. (1999). Neurofibrillary tangles are commonly found in cortical neurons, especially in the entorhinal cortex, as well as in other locations such as pyramidal cells of the hippocampus, the amygdala, the basal forebrain, and the raphe nuclei.
Neurofibrillary tangles can also be found during normal aging of the brain, however, they are found in a significantly higher density in the brain of Alzheimer""s disease patients, and in the brains of patients with other neurodegenerative diseases, such as progressive supranuclear palsy, postencephaltic Parkinson disease, Pick""s disease, amylotrophic lateral sclerosis, etc. Robbins Pathologic Basis of Disease, Cotran et al., 6th ed. (1999), p.1330. Previous studies suggest that, among other things, neurofibrillary tangles may significantly contribute to the cognitive decline associated with the disease and also directly to neuronal cell death.
Ultrastructurally, neurofibrillary tangles are composed predominantly of paired helical filaments (xe2x80x9cPHFxe2x80x9d). A major component of PHF is an abnormally phosphorylated form of a protein called tau and its fragments. Robbins Pathologic Basis of Disease, Cotran et al., 6th ed., W. B. Saunders Company (1999), p.1300.
The tau protein (also referred to as xe2x80x9cnative tauxe2x80x9d) is a microtubule-associated phosphoprotein that stabilizes the cytoskeleton and contributes to determining neuronal shape. See Kosik and Caceres, Cell Sci. Suppl. 14:69-74 (1991). Tau has an apparent molecular weight of about 55 kDa. The protease cathepsin D cleaves tau protein at neutral (cytoplasmic) pH resulting in tau fragmentsxe2x80x94one of which has a molecular weight of approximately 29 kDa (referred to by some authors as xe2x80x9ctau fragmentxe2x80x9d). See, e.g., Bednarski and Lynch, J. Neurochem. 67:1846-1855 (1996); Bednarski and Lynch, NeuroReport 9:2089-2094 (1998). Both the tau protein and 29 kDa tau fragment can be phosphorylated. In a normal brain, the tau protein and tau fragment typically exist in an unphosphorylated, or dephosphorylated state. However, in neurofibrillary tangles, both tau protein and tau fragment can be found in an abnormally phosphorylated state, a hyperphosphorylated state. The 29 kDa tau fragment is a major component of neurofibrillary tangles. Hyperphosphorylation impairs tau protein""s ability to interact with microtubules.
Bednarski E, and Lynch G, J Neurochem 67:1846-55 (1996) cultured hippocampal slices with an inhibitor [N-CBZ-L-phenylalanyl-L-alanine-diazomethyl ketone (ZPAD)] of cathepsins B and L. The authors reported that this resulted in the degradation of high molecular weight isoforms of tau protein and the production of a 29-kDa tau fragment (tau 29).
Bednarski E, and Lynch G, Neuroreport 9:2089-2094 (1998) reported that incubating cultured hippocampal slices with chloroquine or with ZPAD resulted in increases in enzymatically active cathepsin D and the delayed appearance of a 29 kDa fragment of the tau protein. The authors proposed that inactivation of cathepsin L leads to induction of cathepsin D which leads to aberrant tau proteolysis and that such a pathway is likely to play an important role in brain aging.
In addition to the build-up of A-beta and of neurofibrillary tangles, increasing evidence has pointed to a link between lipid metabolism and Alzheimer""s disease. Epidemiological studies found that patients with increased plasma cholesterol levels and cardiovascular diseases have an increased risk of Alzheimer""s disease (Jick, H., et al., Lancet 356:627-631 (2000)). Also, long-term therapy with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors appears to decrease the prevalence of Alzheimer""s disease (Jick, H., et al., Lancet 356:627-631 (2000); Wolozin, B., et al., Arch. Neurol. 57:1439-1443 (2000)).
Consistent with a link to lipid metabolism, in vitro experiments have shown that cholesterol affects the generation and aggregation of beta amyloid (A-beta) (Bodovitz, S., and Klein, W. L., J. Biol. Chem. 271:4436-4440 (1996); Xu, H., et al., Proc. Natl. Acad. Sci. U S A 94:3748-3752 (1997); Howland, D. S., et al., J. Biol. Chem. 273:16576-16582 (1998) ). Transgenic mice fed a high cholesterol diet also developed increased amounts of A-beta deposition (Refolo, L. M., et al., Neurobiol. Dis. 7:321-331 (2000)).
ApoE-mediated transport of cholesterol into lysosomes is a critical step for cells to utilize these sterols, which is of particular importance for mature neurons that mainly rely on extracellular cholesterol (Brown, M. S., and Goldstein, J. L., Annu. Rev. Biochem. 52:223-261 (1983)). Once in the lysosome, cholesterol and other lipids dissociate from ApoE before being utilized by the cell (Brown, M. S., and Goldstein, J. L., Annu. Rev. Biochem. 52:223-261 (1983)).
Changes in cholesterol levels may be involved in certain neurodegenerative diseases. For example, accumulation of insoluble A-beta1-42 has been found in Niemann-Pick type C (NPC) mutant cells (Yamazaki, T., et al., J. Biol. Chem. (2000)(epub ahead of print)). These cells exhibit many pathologic characteristics, one of which is impaired intracellular transport of cholesterol (Millard, E. E., et al., J. Biol. Chem. 275:38445-38451 (2000)). Also, the ApoE4 isoform is a known risk factor for late-onset Alzheimer""s disease.
Inhibition of cholesterol synthesis enhanced the phosphorylation of tau in dissociated cell cultures [ref. in (Sawamura, N., et al., J. Biol. Chem. 57:1439-1443 (2001))]. Likewise, hyperphosphorylation of tau has been demonstrated in cell cultures prepared from NPC mutant mice (Sawamura, N., et al., J. Biol. Chem. 57:1439-1443 (2001)). Gradually developing disturbances in lysosomes, which affect the sorting/trafficking of cholesterol from lysosomes and late endosomes, may, therefore, be contributors to the pathologies associated with neurodegenerative diseases and Alzheimer""s disease.
There has been considerable research into mechanisms underlying neurodegenerative diseases, including Alzheimer""s disease. Many transgenic animal models of Alzheimer""s disease have been developed and used in an attempt to study the mechanisms of Alzheimer""s disease as well as to screen compounds that may ameliorate the conditions of Alzheimer""s disease. However, many in vivo or in vitro models lack some of the important features of Alzheimer""s disease, such as neurofibrillary tangles. Thus, there is an ongoing need to develop a model, especially one useful in vivo or in vitro, that mimics the pathology of neurodegenerative diseases including Alzheimer""s disease and new ways to investigate and combat such conditions. The present invention meets these and other needs.
The present invention provides a model for Alzheimer""s disease and other neurodegenerative diseases. The model of the invention provides brain cells, or brain tissue containing the same, and a method for increasing or decreasing characteristics and changes indicative of neurodegenerative diseases in such cells, especially, the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases and/or any other characteristic or change indicative of neurodegenerative diseases in such cells.
The model of the invention has identified new targets for therapeutic intervention, and new classes of compounds for the treatment of neurodegenerative diseases, and especially, Alzheimer""s disease. For example, the model of the invention has identified the inhibition of tau proteolysis as a new target for therapeutic intervention. As shown herein, cysteine protease inhibitors, and specifically, calpain inhibitors, are capable of inhibiting tau proteolysis and thus the formation of tau fragments. Such inhibitors prevent the formation of neurofibrillary tangles (the formation of which have been induced, according to the model of the invention, by conditions that raise the amount and/or activity of cathepsin D and/or conditions that lower the amount or concentration of cholesterol in the brain tissue).
Accordingly, in one aspect, the invention provides a model of neurodegenerative disease development, such model being a method of increasing the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases, in a suitable brain cell(s), or brain tissue preparation containing the same, the method comprising (1) inducing lysosomal dysfunction and selectively increasing cathepsin D, or, selectively lowering cholesterol, in the brain cell, to levels sufficient to effect the desired changes and (2) culturing the brain cell of part (1) for a period of time sufficient to effect such changes, such changes including the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in such cell relative to the levels found in control cells. In a further embodiment, cathepsin D is selectively increased and also cholesterol is selectively lowered in the brain cells.
In another aspect, the invention provides a method comprising: (a) exposing brain cells, or brain tissue preparation containing the same, to a condition, or contacting brain cells, or brain tissue containing the same, with a compound that inhibits or suppresses lysosomal function, increases cathepsin D, or decreases cholesterol, to a level effective to induce characteristics or indicia of a brain afflicted with a neurodegenerative disease in the cells by the continued exposure thereto; and (b) maintaining the cells for a period of time sufficient to induce such properties or indicia, wherein such properties or indicia include the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases. In a further embodiment, cathepsin D is selectively increased and also cholesterol is selectively decreased in the brain cells, or brain tissue containing the same.
In yet another aspect, the invention provides brain cells, or brain tissue containing the same, that have been exposed to conditions that inhibit or suppress lysosomal function, increase cathepsin D, or, that selectively decrease cholesterol, to a level effective to increase the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in such brain cells, or brain tissue preparations containing the same, compared to such levels in a control. In a further embodiment, the brain cells, or brain tissue containing the same, have been prepared from medium in which both cathepsin D is selectively increased and cholesterol is selectively decreased.
In yet another aspect, the invention provides brain cells, or brain tissue containing the same, that contain (in the media or in the cell), or that have been treated with, a compound that inhibits or suppresses lysosomal function, increases cathepsin D, or that lowers cholesterol in such brain cells, or brain tissue containing the same, to a level effective to increase the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in such brain cells, or brain tissue containing the same, compared to such levels in a control. In a further embodiment, both cathepsin D has been selectively increased and cholesterol levels have been selectively decreased in the cells as a result of such compound. In a preferred embodiment, such compound or its precursor was exogenously administered.
In yet another aspect, the invention provides a screening method comprising: (a) contacting brain cells, or brain tissue containing the same, with a cathepsin D-increasing compound that increases cathepsin D in the brain cells, or with an agent capable of decreasing cholesterol, wherein the change in cathepsin D or cholesterol is sufficient to increase the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in the brain cells, or brain tissue containing the same; (b) contacting the brain cells with an agent; and (c) determining whether the agent modulates the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in the brain cells, as compared to brain cells that are not treated with the agent. In a further embodiment, both cathepsin D is selectively increased and cholesterol is selectively decreased in the brain cells, or brain tissue containing the same, prior to contact with such agent.
In yet another aspect, the invention provides a method of decreasing neurofibrillary tangles, phosphorylated tau and/or tau fragments, or of preventing the formation of the same, in any suitable brain cell, or brain tissue containing the same, that contains, or has been induced to form, such neurofibrillary tangles, phosphorylated tau and/or tau fragments in such brain cell, the method comprising (1) selectively inhibiting the activity of cysteine proteases, and especially of calpain, in the brain cell and (2) culturing the brain cell containing the selectively inhibited protease from part (1) for a period of time sufficient to reduce the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in such cell.
In yet another aspect, the invention provides a method comprising (a) exposing the brain cells, or brain tissue containing the same, to a condition, or contacting the brain cells, or brain tissue containing the same, with a compound, that inhibits the activity of cysteine proteases, or at least of a cysteine protease, and especially calpain, to a level effective to result in a reduction or lessening in the properties or indicia of a brain afflicted with a neurodegenerative disease by the continued exposure to, contact with, or incubation therein, and (b) maintaining such exposure or contact or incubation for a period of time sufficient to reduce such properties or indicia, wherein such properties or indicia include increased amounts of neurofibrillary tangles, phosphorylated tau and/or tau fragments.
In yet another aspect, the invention provides brain cells, or brain tissue containing the same, that have been exposed to a compound or conditions in which cysteine proteases, and especially calpain, in such cells are selectively inhibited, and that lack, or contain a lower amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments as a result of such inhibition.
In yet another aspect, the invention provides brain cells, or brain tissue containing the same, that contain (in the media or in the cell), or that have been treated with, a compound that selectively inhibits cysteine proteases, and especially calpain, in such cells, such brain cells, or brain tissue containing the same, lacking, or containing, a lower amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments as a result of such inhibition.
In yet another aspect, the invention provides a screening method comprising: (a) contacting brain cells, or brain tissue containing the same, with a compound that effectively inhibits the activity of cysteine proteases, and especially calpain, in the brain cells, or brain tissue containing the same, wherein the inhibition of such cysteine proteases, and especially, the inhibition of calpain, decreases, or prevents an increase in, the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells, or brain tissue containing the same; (b) contacting the brain cells, or brain tissue containing the same, with an further agent; and (c) determining whether the agent of part (b) modulates the amount of neurofibrillary tangles, phosphorylated tau and/or tau fragments in the brain cells, or brain tissue containing the same, treated with the agent compared to the brain cells, or brain tissue containing the same, that are not treated with the agent.
In yet another aspect, the invention provides a method of decreasing the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases or of preventing the formation of the same, in any suitable brain cell, or brain tissue containing the same, that contains, or has been induced to form such neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in such brain cell, the method comprising (1) selectively inhibiting the activity of a mitogen activated kinase, and especially of MAP kinase, in the brain cell and (2) culturing the brain cell containing the selectively inhibited kinase from part (1) for a period of time sufficient to reduce the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in such cell.
In yet another aspect, the invention provides a method comprising (a) exposing the brain cells, or brain tissue containing the same, to a condition, or contacting the brain cells, or brain tissue containing the same, with a compound, that inhibits the activity of a mitogen activated kinase, and especially MAP kinase, to a level effective to reduce the properties or indicia of a brain afflicted with a neurodegenerative disease by the continued exposure to, contact with, or incubation therein, and (b) maintaining such exposure or contact or incubation for a period of time sufficient to reduce such properties or indicia, wherein such properties or indicia include one or more of neurofibrillary tangles, phosphorylated tau, and/or tau fragments, the production of cytokines, the release of cytokines, microglia reactions, microglia activations, inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases.
In yet another aspect, the invention provides brain cells, or brain tissue containing the same, that have been exposed to a compound or conditions in which a mitogen activated kinase, and especially MAP kinase, in such cells are selectively inhibited, and that lack, or contain a lower amount of, neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases as a result of such inhibition.
In yet another aspect, the invention provides brain cells, or brain tissue containing the same, that contain (in the media or in the cell), or that have been treated with, a compound that selectively inhibits a mitogen activated kinase, and especially MAP kinase, in such cells, such brain cells, or brain tissue containing the same, lacking, or containing, a lower amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases as a result of such inhibition.
In yet another aspect, the invention provides a screening method comprising: (a) contacting brain cells, or brain tissue containing the same, with a compound that effectively inhibits the activity of a mitogen activated kinase, and especially MAP kinase, in the brain cells, or brain tissue containing the same, wherein the inhibition of such a mitogen activated kinase, and especially, the inhibition of MAP kinases, decreases, or prevents an increase in, the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in the brain cells, or brain tissue containing the same; (b) contacting the brain cells, or brain tissue containing the same, with an further agent; and (c) determining whether the agent of part (b) modulates the amount of neurofibrillary tangles and/or phosphorylated tau and/or tau fragments and/or the production and/or release of cytokines and/or microglia reactions and/or activations and/or inflammation and/or conversion of p35 to p25 and/or the levels and activities of protein kinases in the brain cells, or brain tissue containing the same, treated with the agent compared to the brain cells, or brain tissue containing the same, that are not treated with the agent.
In preferred embodiments of the above models, methods and brain cells, or brain tissue containing the same, xe2x80x9cwild-typexe2x80x9d brain cells from rats or mice, or brain tissue containing the same, apoE-deficient brain cells, or brain tissue containing the same, or apoE4-containing brain cells, or brain tissue containing the same, are used.
In yet another aspect, the invention provides a method for the treatment or prevention of neurodegenerative diseases that are characterized by tau proteolysis, an accumulation of tau fragments, or paired helical filaments, or neurofibrillary tangles, such method comprising the administration of an inhibitor of tau proteolysis to a patient in need of such treatment or prevention.